1. Field of the Invention
The invention relates to cancer diagnosis and treatment, and specifically to the determination of a drug resistance phenotype in neoplastic cells from cancer patients. The invention specifically relates to the separation of chemotherapeutic drug resistant neoplastic cells from drug sensitive neoplastic cells and stromal cells. The invention in particular relates to the identification of genes that are differentially expressed in chemotherapeutic drug resistant neoplastic cells compared with the expression of these genes in drug sensitive neoplastic cells. As part of this identification, the invention provides a pattern of expression from a selected number of identified genes, the expression of which is increased or decreased in chemotherapeutic drug resistant neoplastic cells. The invention provides methods for identifying such genes and expression patterns of such genes and using this information to make clinical decisions on cancer treatment, especially chemotherapeutic drug treatment of cancer patients.
2. Summary of the Related Art
Cancer remains one of the leading causes of death in the United States. Clinically, a broad variety of medical approaches, including surgery, radiation therapy and chemotherapeutic drug therapy are currently being used in the treatment of human cancer (see the textbook CANCER: Principles and Practice of Oncology, 2d Edition, De Vita et al., eds., J. B. Lippincott Company, Philadelphia, Pa., 1985). However, it is recognized that such approaches continue to be limited by a fundamental inability to accurately predict the likelihood of clinically successful outcome, particularly with regard to the sensitivity or resistance of a particular patient""s tumor to a chemotherapeutic agent or combinations of chemotherapeutic agents.
A broad variety of chemotherapeutic agents are used in the treatment of human cancer. These include the plant alkaloids vincristine, vinblastine, vindesine, and VM-26; the antibiotics actinomycin-D, doxorubicin, daunorubicin, mithramycin, mitomycin C and bleomycin; the antimetabolites methotrexate, 5-fluorouracil, 5-fluorodeoxyuridine, 6-mercaptopurine, 6-thioguanine, cytosine arabinoside, 5-aza-cytidine and hydroxyurea; the alkylating agents cyclophosphamide, melphalan, busulfan, CCNU, MeCCNU, BCNU, streptozotocin, chlorambucil, bis-diamminedichloroplatinum, azetidinylbenzoquinone; and the miscellaneous agents dacarbazine, mAMSA and mitoxantrone (DeVita et al., Id.). However, some neoplastic cells become resistant to specific chemotherapeutic agents, in some instances even to multiple chemotherapeutic agents, and some tumors are intrinsically resistant to certain chemotherapeutic agents. Such drug resistance or multiple drug resistance can theoretically arise from expression of genes that confer resistance to the agent, or from lack of expression of genes that make the cells sensitive to a particular anticancer drug. One example of the former type is the multidrug resistance gene, MDR1, which encodes an integral plasma membrane protein termed P-glycoprotein that is an non-specific, energy-dependent efflux pump. (See Roninson (ed)., 1991, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in Biochemical Bases for Multidrug Resistance in Cancer, Academic Press, N.Y., Chapter 11 for reviews). Examples of the latter type include topoisomerase II, the expression of which makes cells sensitive to the anticancer drug etoposide. Decreased expression of this enzyme makes neoplastic cells resistant to this drug. (See Gudkov et al., 1993, Proc. Natl. Acad. Sci. USA 90:3231-3235). Although these are just single examples of the way that modulation of gene expression can influence chemotherapeutic drug sensitivity or resistance in neoplastic cells, these examples demonstrate the diagnostic and prognostic potential for identifying genes the expression of which (or the pattern of gene expression modulation thereof) are involved in mediating the clinical effectiveness of anticancer drug treatment.
Thus, there is a need in this art for developing methods for identifying genes and gene expression patterns that are predictive of the clinical effectiveness of anticancer drug treatment therapies, in order to make more informed decisions for treating individual cancer patients with anticancer drugs having greatest likelihood of producing a positive outcome.
The present invention provides methods identifying genes and gene expression patterns that are predictive of the clinical effectiveness of anticancer drug treatment therapies.
In a first aspect the invention provides a method for separating living neoplastic cells from dead cells and living stromal cells in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with a vital stain or fluorescent dye;
b) contacting the mixed population of cells with a detectably-labeled immunological reagent that specifically binds to neoplastic cells; and
c) selecting the cells in the mixed population of step (b) that are not stained with the vital stain and that bind the immunological reagent.
In a preferred embodiment, the vital stain is propidium iodide. Most preferably, the immunological reagent is a tumor-specific antibody that is detectably labeled with a fluorescent label and the cells are separated by fluorescence activated cell sorting. In certain embodiments, the tumor sample is a solid tumor sample and the mixed cell population is a disaggregated tumor sample. In other embodiments, the tumor sample is a hematopoietic tumor sample and the mixed cell population is a nucleated hematopoietic cell sample.
In a second aspect, the invention provides a method for separating living neoplastic cells that are resistant to a cytotoxic compound from dead cells, living stromal cells and living neoplastic cells that are sensitive to the cytotoxic compound in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with the cytotoxic compound for a time and at a concentration wherein the stromal cells and neoplastic cells that are sensitive to the cytotoxic compound undergo apoptosis;
b) contacting the mixed population of step (a) with a vital stain or fluorescent dye;
c) contacting the mixed population of cells of step (b) with a discrimination compound that specifically binds to apoptotic cells;
d) contacting the mixed cell population of step (c) with a detectably-labeled immunological reagent that specifically binds to the apoptotic cell discrimination compound; and
e) selecting the cells in the mixed population of step (c) that are not stained with the vital stain and that do not bind the immunological reagent.
In a preferred embodiment, the vital stain is propidium iodide. Most preferably, the apoptosis discrimination reagent is Annexin V and the immunological reagent is an Annexin V-specific antibody that is detectably labeled with a fluorescent label , wherein the cells are separated by fluorescence activated cell sorting. In preferred embodiments, the mixed population is contacted with the cytotoxic compound under in vitro cell culture conditions whereby the cells cannot attach to a solid substrate. In certain embodiments, the tumor sample is a solid tumor sample and the mixed cell population is a disaggregated tumor sample. In other embodiments, the tumor sample is a hematopoietic tumor sample and the mixed cell population is a nucleated hematopoietic cell sample.
In yet a third aspect, the invention provides a method for detecting a gene expression profile of living neoplastic cells that are resistant to a cytotoxic compound and distinguishing such a profile from the gene expression profile of living neoplastic cells that are sensitive to the cytotoxic compound in a mixed population of cells from a tumor sample, the method comprising the steps of:
a) contacting the mixed population of cells with the cytotoxic compound for a time and at a concentration wherein the neoplastic cells that are sensitive to the cytotoxic compound undergo apoptosis;
b) contacting the mixed population of step (a) with a vital stain or fluorescent dye;
c) contacting the mixed population of cells of step (b) with a discrimination compound that specifically binds to apoptotic cells;
d) contacting the mixed cell population of step (c) with a detectably-labeled immunological reagent that specifically binds to the apoptotic cell discrimination compound; and
e) separating the cells in the mixed population of step (d) that are not stained with the vital stain from the cells that are stained with the vital stain;
f) separating the cells in the mixed population of step (e) that are not stained with the vital stain and that do not bind the immunological reagent from the cells in the mixed population of step (c) that are not stained with the vital stain and that do bind the immunological reagent;
g) isolating cellular RNA from the each of the separated cells selected in step (f);
h) preparing detectably-labeled cDNA from the cellular RNA isolated in step (g);
i) hybridizing each of the CDNA preparations prepared in step (h) to a gene array comprising at least 4000 eukaryotic genes;
j) detecting a pattern of gene expression for hybridization of each of the cDNA preparations prepared from the mRNA isolated from the cells selected in step (f); and
k) comparing the pattern of gene expression detected in step (j) from ahybridization of the microarray with cDNA from cells that are not stained with the vital stain and that do not bind the immunological reagent with a pattern of gene expression obtained by hybridizing cDNA prepared from cells that are not stained with the vital stain and that do bind the immunological reagent.
In a preferred embodiment, the vital stain is propidium iodide. Most preferably, the apoptosis discrimination reagent is Annexin V and the immunological reagent is an Annexin V-specific antibody that is detectably labeled with a fluorescent label , wherein the cells are separated by fluorescence activated cell sorting. In preferred embodiments, the mixed population is contacted with the cytotoxic compound under in vitro cell culture conditions whereby the cells cannot attach to a solid substrate. In certain embodiments, the tumor sample is a solid tumor sample and the mixed cell population is a disaggregated tumor sample. In other embodiments, the tumor sample is a hematopoietic tumor sample and the mixed cell population is a nucleated hematopoietic cell sample.
In another aspect, the invention provides a diagnostic assay for characterizing tumors and neoplastic cells, particularly human neoplastic cells, wherein cytotoxic drug resistant, and most preferably chemotherapeutic drug resistant neoplastic cells, by the differential expression of genes and patterns of genes whereby the drug resistant phenotype is associated with, identified by and can be diagnosed on the basis thereof. This diagnostic assay comprises detecting, qualitatively or preferably quantitatively, the expression levels of a gene or a plurality of genes comprising a pattern of expression of genes and making a diagnosis of drug resistance on the basis of this expression pattern of a gene or plurality of genes. In a preferred embodiment, the invention provides methods for identifying a gene or a plurality of genes showing differential gene expression in drug resistant neoplastic cells. In other preferred embodiments, the invention provides methods for detecting expression of a gene or a plurality of genes comprising a pattern of gene expression in drug resistant neoplastic cells. In still other embodiments, the invention provides these methods for a multiplicity of chemotherapeutic drugs to determine drugs for which a patient""s tumor is not resistant or shows a minimal level of resistance.
In another embodiment, the invention provides a starting point for in vitro drug screening and rational design of pharmaceutical products that are more effective antineoplastic agents. By identifying a pattern of differential gene expression related to drug resistance, strategies can be developed for creating pharmaceutical products that are improved chemotherapeutic agents to more effectively treat neoplastic disease.
It is an advantage of the methods of this invention that pure neoplastic cell populations from solid and hematopoietic tumors, both malignant and benign, can be obtained separated from stromal cells, infiltrating non-neoplastic hematopoietic cells and other tumor components. This feature of the inventive methods are advantageous because the presence of such contaminating, non-neoplastic cells in tumor sample preparations confounds analyses directed at detecting neoplastic cell-specific properties, such as patterns of gene expression as disclosed herein. It is also an advantage of the present inventive methods that drug-resistant and drug-sensitive neoplastic cells can be separated from pure neoplastic cell populations. As a result, RNA preparations specific for drug-resistant and drug-sensitive neoplastic cells are obtained that can be used to identify genes, and patterns of genes, that are differentially expressed in drug-resistant and drug-sensitive neoplastic cells. In addition, the methods of the invention as provided permit drug-resistant and drug-sensitive neoplastic cells to be simultaneously treated with cytotoxic drugs in the same mixed cell culture, thereby avoiding experimental variability.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.